Method and system for analysing fluorospot assays

ABSTRACT

Disclosed is a method for analysing FluoroSpot assays. The method comprises illuminating a well of an assay plate with at least one excitation light, capturing at least one image of the well, in raw image format, for each excitation light, generating a model of analyte release distribution in the well for each excitation light, and clustering a plurality of co-positioned fluorospots as a multiple secretion fluorospot, wherein the clustering is performed for all generated models, and wherein the clustering determines at least one multiple secretion fluorospot. The generation of the model of analyte release distribution for a given excitation light comprises deconvolving the captured at least one image of the well to estimate a pre-diffusion analyte distribution, and detecting potential analyte release sites based on local maxima therein.

TECHNICAL FIELD

The present disclosure relates generally to investigative techniquessuch as assays; and more specifically, to methods and systems foranalysing fluorospot assays. The present disclosure also relates to acomputer implementable program operable to execute the aforesaid methodon the aforesaid apparatus.

BACKGROUND

Typically, investigative procedures, such as assays, are employed infields of medicine, molecular biology, and so forth. For example, assaysare widely utilized in immunology for determining rate of activation ofcells in response to vaccines, infections, allergens, etc. Assaysinvolving antibodies as key components are typically referred to asimmunoassays. Examples of immunoassays include, but are not limited to,Enzyme-Linked Immunosorbent Assay (ELISA), Enzyme-Linked ImmunoSpot(ELISPOT) assay, fluorospot assay.

Generally, the immunoassays include detection and quantitation ofanalytes in liquid samples or the monitoring of secreted analytes (forexample, cytokines and immunoglobulins) by cells under observation. Forexample, in a fluorospot assay, cytokine-specific capture antibodies maybe added to an assay plate having wells therein. Thereafter, cells maybe added to the wells in presence or absence of activating stimuli andthe assay plate may be incubated to allow for cytokine secretion by thecells. The secreted cytokines may be captured by the immobilizedcytokine-specific capture antibodies at the bottom of wells. Thereafter,the cells may be removed and the wells may be washed and added withcytokine-specific detection antibodies and fluorophore conjugates.Finally, secretory footprints (or spots) of the secreted cytokines arecaptured by way of imaging, and such images are analysed for identifyingmultiple analytes, counting number of cells secreting the analytes, andthe like.

However, there exist a number of limitations associated with analysis ofsuch assays. Firstly, the cells under observation secrete the analytesin different quantities, thereby creating spots of varying sizes andintensities. Therefore, over a period of time, such secretion may leadto different spatial intensity profiles among spots of the analytes.Further, in many instances, the captured images may be saturated (orover exposed) by imaging equipment, and the true spatial intensityprofiles of the spots are not clearly visible. Often, the cells areclosely located to each other, thereby, leading to overlap of the spotsin the captured images. Currently existing analytic techniques are notsufficiently developed to accurately determine secretion intensities ofindividual cells, and distinguish individual spots. The saturation ofcaptured images further exacerbates problems associated withdistinguishing individual spots from overlapped spots. Secondly, thecurrently existing techniques are unable to accurately determine centresof the spots. Specifically, the centres of spots emit strongerfluorescent signals as compared to other regions of the spots, thereby,facilitating identification of multiple secretions from a single cell.However, due to existing limitations in accurate determination of thecentres of spots, identification of multiple secretions from the cellsis difficult, and prone to errors. Thirdly, equipment employed forcurrently existing techniques introduces problems in the analysis ofassays, such as compression of the captured images by the imagingequipment, inaccurate capture of images (such as shift between theimages), blurring in the captured images on account of vibrations in theequipment, chromatic aberration and so forth.

Fourthly, the equipment employed for conventional techniques fail toeffectively measure the concentration of analytes in one or more wellsof the array. Furthermore, it deploys duplication of tests for measuringthe concentration of the analytes, thereby, increasing time, cost andcomplexity of the process. Nonetheless, the entire process results in areduction of efficacy in determining the potential analyte releasingcells and the concentration of the analyte in the well.

Therefore, in light of the foregoing discussion, there exists a need toovercome the aforementioned drawbacks associated with analysis ofassays.

SUMMARY

The present disclosure seeks to provide a method for analysingfluorospot assays. The present disclosure also seeks to provide a systemfor analysing fluorospot assays. The present disclosure seeks to providea solution to the existing problems of inaccurate determination ofsecretion intensities of individual cells, errors in distinguishingindividual spots and measuring concentration of the analytes inconventional assay analysis techniques. An aim of the present disclosureis to provide a solution that overcomes at least partially the problemsencountered in prior art, and provides an efficient, robust, and easy toimplement assay analysis technique.

In a first aspect, an embodiment of the present disclosure provides amethod for analysing fluorospot assays, the method comprising:

-   -   (i) illuminating a well of an assay plate having plurality of        wells, with a plurality of excitation lights, wherein at a given        time, the well of the assay plate is illuminated by only one        excitation light;    -   (ii) capturing at least one image of the well, in raw image        format, for each of the at least one excitation light;    -   (iii) generating a model of analyte release distribution in the        well for each of the at least one excitation light, wherein the        generation of the model of analyte release distribution in the        well for a given excitation        -   (a) deconvolving the captured at least one image of the well            for the given excitation light to estimate a pre-diffusion            analyte distribution; and        -   (b) detecting at least one potential analyte release site            based on local maxima in the pre-diffusion analyte            distribution; and    -   (iv) for the plurality of excitation lights employed to        illuminate the well of the assay plate, clustering a plurality        of co-positioned fluorospots as a multiple secretion fluorospot,        wherein the clustering is performed for all generated models of        analyte release distribution, and wherein the clustering        determines at least one multiple secretion fluorospot,        characterized in that the method includes:        generating the model of analyte release distribution in the well        for each of the plurality of excitation light comprises        optimizing the pre-diffusion analyte distribution based on the        detected at least one potential release site; and modifying the        model of analyte release distribution in the well to analyse at        least one fluorospot therein, wherein optimizing the        pre-diffusion analyte distribution, based on the detected at        least one potential analyte release site, is implemented        iteratively at least once, and comprises employing at least one        of: alternating direction methods, multiplicative update rules,        forward-backward proximal gradient algorithms.

The present disclosure is of advantage in that it efficiently detectsand quantitates the analytes secreted by the cells in liquid samples.

Embodiments of the disclosure are advantageous in terms of providing amethod that conforms to an advanced and highly efficient and robustanalytical technique. Moreover, the embodiments of the disclosure areadvantageous in terms of an automatic sensing mechanism for detectingthe high fluorescent intensity and correlating the fluorescent intensitywith the concentration of the analyte in the well(s). Beneficially, theanalyte distribution and/or concentration measurement require minimal orno manual intervention, thereby reducing the cost of operation of theassay.

Optionally, the plurality of excitation lights for illuminating the wellof the assay plate depends on the at least one fluorophore-detectedanalyte in the spatially defined area of the well.

Optionally, deconvolving the captured at least one image of the wellcomprises implementing at least one of: sparsity-promotingregularization, multiplicative update rules, forward-backward proximalgradient algorithms.

Optionally, modifying the model of analyte release distribution in thewell is based on at least one user-selectable parameter, wherein the atleast one parameter is selected from a group comprising:fluorospot/fluorescent intensity, fluorospot size, estimated amount ofanalyte release, fluorospot colour, area of interest in the well.

Optionally, clustering the plurality of co-positioned fluorospotsemploys a distance-based hierarchical clustering algorithm.

Optionally, the method further comprises displaying a resultant model ofanalyte release distribution in the well, wherein the model of analyterelease distribution, based on the detected at least one potentialanalyte release site, is implemented iteratively at least once, andcomprises employing at least one of: alternating direction methods,multiplicative update rules, forward-backward proximal gradientalgorithms, wherein the resultant model comprises the detected at leastone fluorospot, and wherein single secretion fluorospots and multiplesecretion fluorospots are represented.

In a second aspect, an embodiment of the present disclosure provides asystem for analysing fluorospot assays, the system comprising:

-   -   (i) a light source arrangement configured to generate a        plurality of excitation lights, wherein at a given time, the        light source arrangement generates only one excitation light;    -   (ii) a collimation arrangement positioned on an optical path of        a generated excitation light, wherein the collimation        arrangement is configured to collimate the generated excitation        light;    -   (iii) a multiband beam splitter positioned on an optical path of        the collimated excitation light, the multiband beam splitter        being supported by a support arrangement, wherein the multiband        beam splitter is configured to        -   (a) reflect the collimated excitation light onto an assay            plate for illuminating a well of the assay plate having a            plurality of wells;        -   (b) receive a reflection of the excitation light and an            emitted light from the assay plate; and        -   (c) reflect a portion of the reflected excitation light            whilst transmitting a remaining portion of the reflected            excitation light and the emitted light to a filtering            arrangement;    -   (iv) the filtering arrangement comprising a filter wheel and at        least one emission filter, wherein the filter wheel is        configured to accommodate the at least one emission filter        therein, and wherein the at least one emission filter is        configured to        -   (d) remove the remaining portion of the reflected excitation            light received from the multiband beam splitter; and        -   (e) filter the emitted light whilst transmitting the emitted            light to an optic arrangement;    -   (v) the optical arrangement configured to direct the filtered        emitted light onto an imaging device;    -   (vi) the imaging device configured to capture at least one image        of the well, in raw image format, for each of the plurality of        excitation lights; and    -   (vii) a processing module coupled to the imaging device, wherein        the processing module is configured to        -   (f) generate a model of analyte release distribution in the            well for each of the plurality of excitation lights, wherein            optimizing the pre-diffusion analyte distribution, based on            the detected at least one potential analyte release site, is            implemented iteratively at least once, and comprises            employing at least one of: alternating direction methods,            multiplicative update rules, forward-backward proximal            gradient algorithms; and        -   (g) for the plurality of excitation lights employed to            illuminate the well of the assay plate, cluster a plurality            of co-positioned fluorospots as a multiple secretion            fluorospot, wherein the clustering is performed for all            generated models of analyte release distribution, and            wherein the clustering determines at least one multiple            secretion fluorospot.

Optionally, the system further comprises at least one excitation filterpositioned on an optical path of the collimated excitation light, andwherein the at least one excitation filter is configured to filter thecollimated excitation light.

Optionally, the light source arrangement comprises at least one lightsource positioned on an actuator arrangement, wherein the at least onelight source is implemented by way of at least one of: Light EmittingDiode, halogen light source, xenon light source, laser light source.

Optionally, the multiband beam splitter is implemented by way of atleast one of: a transparent mirror, a semitransparent mirror, a prism, adichroic mirror.

Optionally, the optical arrangement comprises at least one of:telecentric lens, macro lens.

Optionally, the support arrangement is a filter cube.

Optionally, the system further comprises a visualization module coupledto the imaging device and the processing module, wherein thevisualization module comprises a user interface rendered on a computingdevice associated with a user, and wherein the visualization module isconfigured to perform at least of: display the captured at least oneimage for each of the at least one excitation light, display thegenerated model of analyte release distribution in the well for each ofthe at least one excitation light, receive user input to controloperation of the processing module, display a resultant model of analyterelease distribution in the well.

Embodiments of the present disclosure substantially eliminate or atleast partially address the aforementioned problems in the prior art,and enables accurate and reliable assay analysis.

Additional aspects, advantages, features and objects of the presentdisclosure would be made apparent from the drawings and the detaileddescription of the illustrative embodiments construed in conjunctionwith the appended claims that follow.

It will be appreciated that features of the present disclosure aresusceptible to being combined in various combinations without departingfrom the scope of the present disclosure as defined by the appendedclaims.

A better understanding of the present invention may be obtained throughthe following examples which are set forth to illustrate, but are not tobe construed as limiting the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The summary above, as well as the following detailed description ofillustrative embodiments, is better understood when read in conjunctionwith the appended drawings. For the purpose of illustrating the presentdisclosure, exemplary constructions of the disclosure are shown in thedrawings. However, the present disclosure is not limited to specificmethods and instrumentalities disclosed herein. Moreover, those in theart will understand that the drawings are not to scale. Whereverpossible, like elements have been indicated by identical numbers.

Embodiments of the present disclosure will now be described, by way ofexample only, with reference to the following diagrams wherein:

FIG. 1 is a schematic illustration of a system for analysing FluoroSpotassays, in accordance with an embodiment of the present disclosure;

FIGS. 2A, 2B and 2C are schematic illustrations of images of a generatedmodel of a first analyte release distribution, a generated model of asecond analyte release distribution, and a resultant model of analyterelease distribution in a well, respectively, in accordance with anembodiment of the present disclosure;

FIG. 3 illustrates steps of a method for analysing fluorospot assays, inaccordance with an embodiment of the present disclosure;

FIG. 4 is a schematic illustration of a system for determining theconcentration of at least one fluorophore-detected analyte in aspatially defined area of the well, in accordance with an embodiment ofthe present disclosure; and

FIG. 5 illustrates steps of a method for determining the concentrationof at least one fluorophore-detected analyte in a spatially defined areaof the well, in accordance with an embodiment of the present disclosure.

In the accompanying drawings, an underlined number is employed torepresent an item over which the underlined number is positioned or anitem to which the underlined number is adjacent. A non-underlined numberrelates to an item identified by a line linking the non-underlinednumber to the item. When a number is non-underlined and accompanied byan associated arrow, the non-underlined number is used to identify ageneral item at which the arrow is pointing.

DETAILED DESCRIPTION OF EMBODIMENTS

It is appreciated that certain features of the invention, which are forclarity described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely variousfeatures of the invention, which are for brevity, described in thecontext of a single embodiment, may also be provided separately and/orin any suitable sub-combination.

The following detailed description illustrates embodiments of thepresent disclosure and ways in which they can be implemented. Althoughsome modes of carrying out the present disclosure have been disclosed,those skilled in the art would recognize that other embodiments forcarrying out or practicing the present disclosure are also possible.

In one aspect, an embodiment of the present disclosure provides a methodfor analysing fluorospot assays, the method comprising:

(i) illuminating a well of an assay plate having plurality of wells,with a plurality of excitation lights, wherein at a given time, the wellof the assay plate is illuminated by only one excitation light;

(ii) capturing at least one image of the well, in raw image format, foreach of the at least one excitation light;

(iii) generating a model of analyte release distribution in the well foreach of the at least one excitation light, wherein the generation of themodel of analyte release distribution in the well for a given excitation

-   -   (a) deconvolving the captured at least one image of the well for        the given excitation light to estimate a pre-diffusion analyte        distribution; and    -   (b) detecting at least one potential analyte release site based        on local maxima in the pre-diffusion analyte distribution; and        (iv) for the plurality of excitation lights employed to        illuminate the well of the assay plate, clustering a plurality        of co-positioned fluorospots as a multiple secretion fluorospot,        wherein the clustering is performed for all generated models of        analyte release distribution, and wherein the clustering        determines at least one multiple secretion fluorospot,        characterized in that the method includes:        generating the model of analyte release distribution in the well        for each of the plurality of excitation lights comprises        optimizing the pre-diffusion analyte distribution based on the        detected at least one potential release site; and modifying the        model of analyte release distribution in the well to analyse at        least one fluorospot therein.

In another aspect, an embodiment of the present disclosure provides asystem for analysing fluorospot assays, the system comprising:

(i) a light source arrangement configured to generate at least oneexcitation light, wherein at a given time, the light source arrangementgenerates only one excitation light;

(ii) a collimation arrangement positioned on an optical path of agenerated excitation light, wherein the collimation arrangement isconfigured to collimate the generated excitation light;

(iii) a multiband beam splitter positioned on an optical path of thecollimated excitation light, the multiband beam splitter being supportedby a support arrangement, wherein the multiband beam splitter isconfigured to

-   -   (a) reflect the collimated excitation light onto an assay plate        for illuminating a well of the assay plate having a plurality of        wells;    -   (b) receive a reflection of the excitation light and an emitted        light from the assay plate; and (c) reflect a portion of the        reflected excitation light whilst transmitting a remaining        portion of the reflected excitation light and the emitted light        to a filtering arrangement;        (iv) the filtering arrangement comprising a filter wheel and at        least one emission filter, wherein the filter wheel is        configured to accommodate the at least one emission filter        therein, and wherein the at least one emission filter is        configured to    -   (d) remove the remaining portion of the reflected excitation        light received from the multiband beam splitter; and    -   (e) filter the emitted light whilst transmitting the emitted        light to an optic arrangement;        (v) the optical arrangement configured to direct the filtered        emitted light onto an imaging device;        (vi) the imaging device configured to capture at least one image        of the well, in raw image format, for each of the at least one        excitation light; and        (vii) a processing module coupled to the imaging device, wherein        the processing module is configured to    -   (f) generate a model of analyte release distribution in the well        for each of the at least one excitation light; and    -   (g) cluster a plurality of co-positioned fluorospots as a        multiple secretion fluorospot, wherein the clustering is        performed for all generated models of analyte release        distribution, and wherein the clustering determines at least one        multiple secretion fluorospot.

The present disclosure provides a method and a system for analysingfluorescent assays. The described method and system are simple, robust,and easy to implement as compared to conventional techniques andequipment for analysis of assays. Beneficially, the described methodaccurately determines secretion intensities of cells by analysingdifferent spatial intensity profiles among spots of the secretedanalytes, and distinguishes individual spots. Further, the describedmethod accurately determines centres of the spots, thereby, facilitatingaccurate identification of multiple secretions from a single cell.Furthermore, the method minimizes the risk of saturation of sensors byemploying lower exposure to strong spots with high fluorescentintensities. Additionally, the system effectively measures theconcentration of the analytes using a fully-automated sensor andcalibration instrument. Moreover, the system described herein isinexpensive, easy to use, reliable, time efficient and reduces errors onaccount of manual inspection, compression of captured images by imagingequipment, blurring in the captured images, due to moving or vibratingcomponents and/or imprecision in focus, wrong calibration, temporal orinherent noise and so forth.

In an embodiment, the term ‘assay’ used herein relates tofluorescence-based assays. Specifically, such fluorescence-based assays(for example, fluorospot assays) utilize principles of fluorescence inorder to investigate cells, such as human cells, animal cells, and soforth. More specifically, the fluorescence-based assays enablesimultaneous analysis of different analytes secreted by the cells underinvestigation, by employing fluorophore-labeled detection reagents todistinctly identify the different analytes. Therefore, secretoryfootprints (or fluorospots) of the different analytes are distinct fromeach other. Furthermore, fluorescence-based assays enable measuring theconcentration of the analytes secreted by cells based on the fluorescentintensity of the analytes in a sample, specifically a liquid solution.According to an embodiment of the present disclosure, the term ‘analyte’used herein relates to a substance that is analysed by implementingassays, such as a flurospot assay.

In an embodiment of the present disclosure, the term ‘fluorospot assay’used herein relates to immunoassays, enumerating analytes secreted bycells. Specifically, the fluorospot assay relates to a method ofdetection of analytes secreted by cells, such as human cells, animalcells and so forth. Furthermore, in an embodiment, the term ‘fluorospot’used herein relates to secretory footprints of an analyte. Specifically,the secretory footprint of an analyte is sensitive to a wavelength of anexcitation light. More specifically, the secretory footprint of theanalyte is captured by an imaging device for detection of the analyte.

The method for analysing fluorospot assays comprises illuminating a wellof an assay plate having plurality of wells, with at least oneexcitation light, wherein at a given time, the well of the assay plateis illuminated by only one excitation light. Specifically, the pluralityof wells of the assay plate (commonly referred to as a microtiter plate,microplate, microwell plate, and the like) include at least onefluorophore-detected analyte (hereinafter referred to as ‘at least oneanalyte’). Further, upon illumination by the at least one excitationlight, the at least one analyte may emit fluorescence (known as ‘atleast one emitted light’). Furthermore, at least one analyte secreted bythe cell may be bound to a mixture of tag-labelled and biotinylatedanalyte-specific detection antibodies. Moreover, the mixture may befurther bound to detection reagents conjugated to differentfluorophores. In an embodiment, a wavelength of the at least one emittedlight may be greater than a wavelength of the at least one excitationlight. It is to be understood that although the method described hereinrelates to analysis of one well of the assay plate, the method may beimplemented to analyse more than one well of the assay plate.

In an embodiment, the plurality of excitation lights for illuminatingthe well of the assay plate depends on the at least onefluorophore-detected analyte in the spatially defined area of the well.Since analytes may be detected using distinct fluorophore detection, adistinct excitation light may be employed to illuminate and analyse adistinct analyte. For example, a well of an assay plate including twofluorophore-detected cytokines ‘A’ and ‘B’ may be illuminated with twoexcitation lights ‘L1’ and ‘L2’. In such example, wavelength ofexcitation light L1 may match wavelength of a fluorophore employed tolabel the cytokine A, and wavelength of excitation light L2 may matchwavelength of a fluorophore employed to label the cytokine B.

Further, the method comprises capturing at least one image of the well,in raw image format, for each of the at least one excitation light.Specifically, the captured at least one image may be constituted usingthe at least one emitted light that is emitted from the at least oneanalyte when the well is illuminated by the at least one excitationlight. More specifically, for a given excitation light, the captured atleast one image corresponding thereto, visually represent secretoryfootprints (or fluorospots) of an analyte that is sensitive towavelength of the given excitation light. It is to be understood, that anumber (or a count) of the captured at least one image for differentexcitation lights may or may not be equal. Referring to theaforementioned example, 20 images of the well may be captured for theexcitation light L1, and 30 images of the well may be captured for theexcitation light L2. Therefore, the captured 20 images visuallyrepresent fluorospots of the cytokine A, and the captured 30 imagesvisually represent fluorospots of the cytokine B.

According to an embodiment of the present disclosure, the at least oneimage of the well is indicative of spatially located analyte sources andtemporal distribution. Specifically, the term ‘spatially located analytesources’ relates to a spatial distribution of the fluorospots of the atleast one analyte in an image of the well. Further, the term ‘temporaldistribution’ relates to fluorospots of the at least one analyte at acertain time in the captured at least one image of the well.Specifically, the captured at least one image represents temporaldistribution in the fluorospots of the at least one analyte. In anexample, the captured at least one image of the well may provide anindication of the temporal distribution of an analyte based on thespatial distribution of the analyte and dispersion of the analyte in thewell. In another example, the captured at least one image of the wellmay not comprise analyte in the well, and is captured for determiningall well coordinates, depth of well, centre of well and the exactposition of the well borders.

It will be appreciated that the captured at least one image of the wellfor each of the plurality of excitation lights are in raw image format.Beneficially, images captured in raw image format are in unprocessedform and are therefore free from colourspace-dependent encoding.Optionally, the captured at least one image of the well for each of theplurality of excitation lights have image file formats, including, butnot limited to, Joint Photographic Experts Group format, Tagged ImageFile Format, Portable Network Graphics format, and Graphics InterchangeFormat.

Furthermore, capturing at least one image of the well is performed byway of imaging. Specifically, the term ‘imaging’ relates torepresentation or creation of an object (or a scene) by recording light(or other electromagnetic radiations) emanating from the object, bymeans of emission or reflection. More Specifically, a real image isproduced on an image-sensing surface inside an imaging device during atimed exposure. Typically, the image-sensing surface comprises an arrayof pixels arranged in color-filter units (or cells) for generating red,blue, green and white image signals.

It will be appreciated that imaging is performed by using any one of: ahigh-dynamic-range imaging device, a low-dynamic-range imaging device, adigital camera. Embodiments of the disclosure employ ahigh-dynamic-range (indicated by ‘HDR’ hereafter) imaging device.Specifically, the HDR imaging employs combining two or more images toproduce a greater range of luminance in a final image as compared tostandard digital imaging techniques. More specifically, HDR imagingemploys taking several images with different exposures and then mergingthe images into a single HDR image. In an example, the differentexposures may be −1 EV, 0 EV and +1 EV. Beneficially, HDR imagingemploys a suitable HDR software that enables high sensitivity, excellentmeasuring range, higher luminance, minimized risk of saturation ofsensor, tone mapping, much greater range of colors and brightness, imagealignment, filtering random noise, and so forth.

Further, the method comprises generating a model of analyte releasedistribution in the well for each of the at least one excitation light,wherein the generation of the model of analyte release distribution inthe well for a given excitation light comprises deconvolving thecaptured at least one image of the well for the given excitation lightto estimate a pre-diffusion analyte distribution, wherein suchdeconvolution is performed prior to local diffusion of at least oneanalyte, and detecting at least one potential analyte release site basedon local maxima in the pre-diffusion analyte distribution. In anembodiment, the generated model for each of the plurality of excitationlights is a mathematical function. Furthermore, the mathematicalfunction may be a three-dimensional function, wherein the mathematicalfunction may represent the spatial distribution of the analyte providedon a two-dimensional plane (for example, an x-y plane) and a thirddimension thereof may be indicative of the temporal distribution ofanalyte. Subsequently, the third dimension of the mathematical functionmay be projected onto the two-dimensional plane to provide atwo-dimensional mathematical function. Moreover, the two-dimensionalmathematical function may be similar to a two-dimensional image.Therefore, the generated model for each of the plurality of excitationlights is a mathematical function representative of the analytedistribution in the well (specifically, of secretory footprints orfluorospots of the at least one analyte) for each of the at least oneexcitation light. Further, it is to be understood that a wavelength offluorospots representing different analytes is different. Specifically,each generated model of analyte release distribution representsdistribution of a distinct analyte in the well, and such distribution isdetected by way of illuminating the well with a specific excitationlight, and capturing the at least one image of the well, in raw imageformat, for the specific excitation light to generate the model ofanalyte release distribution for the distinct analyte. For example, twomodels (G1 and G2) of analyte release distribution in a well for twoexcitation lights (L3 and L4) may be generated. In such example, themodel G1 may represent distribution of analyte A1 in the well and themodel G2 may represent distribution of analyte A2 in the well.Therefore, a wavelength of fluorospots of analyte A1 in the model G1 isdifferent from a wavelength of fluorospots of analyte A2 in the modelG2.

Optionally, the method further comprises generating an X-Y tableassociated with the X-Y plane of the assay plate and obtaining thefluorescent intensity at the at least one spatially defined fluorospotbased on the generated X-Y table. The term “X-Y table” relates tomotorized linear slides with a linear motion, horizontal or vertical.Specifically, the X-Y table provides horizontal motion, i.e. along an Xand Y axis of an XY plane, for an automated machinery. The XY plane ofthe assay plate includes a plurality of wells separated by a predefineddistance. In an embodiment, the method of the present disclosure enablesdetection of data corresponding to the well coordinates, depth of well,centre of well and the exact position of the well borders. Optionally,such data may also include fluorospot size, peripheral distance betweenadjacent wells, distance from the center of at least two adjacent wells.In another embodiment, the X-Y table associated with the X-Y plane ofthe assay plate enables measuring the distance from the center of atleast two adjacent wells. More optionally, the X-Y table is generated byemploying a calibration algorithm. Specifically, the calibrationalgorithm is developed to calibrate the X-Y table. More specifically,the calibration algorithm is developed to calibrate and re-calibrate theX-Y table upon multiple series of rounds. In an embodiment, thecalibration algorithm is programmed to visit every fluorospot on the XYplane of the assay plate. In yet another embodiment, the datacorresponding to the well coordinates, depth of well, centre of well andthe exact position of the well borders, fluorospot size, peripheraldistance between adjacent wells, distance between the centers of twoadjacent wells and so on, are predefined/preset to fully automate theprocess. Alternatively, additionally, the calibration algorithm isaccessible for modification by a user by way of receiving manual inputsfrom the user of the system. Beneficially, fully (or semi) automatedsystem provides benefits of time-efficiency and reduction in wrongcalibration. In an embodiment, deconvolution of the captured at leastone image of the well is an image processing technique that isimplemented in order to sharpen the captured at least one image of thewell for each of the at least one excitation light, by removing blurringcaused by the diffusion and imaging equipment. Specifically,deconvolution may be implemented in order to determine an image amongthe captured at least one image for the given excitation light, whereinthe determined image represents the pre-diffusion analyte distributionfor the given excitation light. Such determined image is considered tobe one which is most closely representative of the pre-diffusion analytedistribution among the captured at least one image for the givenexcitation light. More specifically, such deconvolution is performed onthe captured at least one image for each of the at least one excitationlight, thereby, taking into account both the spatially located analytesources and temporal distribution represented by the at least one image.Further in an embodiment, an input of such deconvolution is the capturedat least one image for the at least one excitation light, and an outputof such deconvolution step is an image representing the pre-diffusionanalyte distribution for each of the given excitation light. In anotherembodiment, an input of such deconvolution is the mathematical model ofthe analyte release distribution and the output of such deconvolutionstep is an image representing the pre-diffusion analyte distributionprovided by multidimensional deconvolution.

According to an embodiment, deconvolving the captured at least one imageof the well comprises implementing at least one of: sparsity-promotingregularization, multiplicative update rules, forward-backward proximalgradient algorithms. Specifically, at least one of the aforementionedalgorithms may be employed in order to estimate the pre-diffusionanalyte distribution for each of the at least one excitation light. Forexample, sparsity-promoting regularization may be implemented in form ofI1-regularization or group-sparsity regularization based on(non-squared) form of I2-regularization over the captured at least oneimage for the given excitation light, for implementing regularizationamong the at least one image during such deconvolution. In anotherexample, multiplicative update rules and/or forward-backward proximalgradient algorithms may facilitate optimization of variables during suchdeconvolution. Examples of forward-backward proximal gradient algorithmsinclude, but are not limited to, iterative shrinkage-thresholdingalgorithms (ISTA), and the accelerated proximal gradient algorithms(FISTA).

Optionally, the method further comprises identifying a centre of thewell for determining the spatially defined fluorospot. Specifically,local maxima at the center of each well is known as a calibration pointor a hard spot or a spatially defined fluorospot. In an embodiment,detecting the at least one potential analyte release site based on localmaxima in the pre-diffusion analyte distribution, may be implementedusing conventional mathematical formulae and image processing functionsfor determining local maxima and evaluating the image representing thepre-diffusion analyte distribution, respectively.

In an embodiment, generation of the model of analyte releasedistribution in the well for a given excitation light comprisesoptimizing the pre-diffusion analyte distribution based on the detectedat least one potential release site to generate the model of analyterelease distribution in the well for the given excitation light.Optionally, optimizing the pre-diffusion analyte distribution relates torefitting the image representing the pre-diffusion analyte distributionbased on the detected at least one potential analyte release site.Specifically, in such optimization, a constraint is added to the imagerepresenting the pre-diffusion analyte distribution, wherein theconstraint is that analytes may be released only from the detected atleast one potential analyte release site. According to an embodiment,optimizing the pre-diffusion analyte distribution based on the detectedat least one potential analyte release site, is implemented iterativelyat least once, and comprises employing at least one of: alternatingdirection methods, multiplicative update rules, forward-backwardproximal gradient algorithms. Examples of the alternating directionmethods include, but are not limited to, Douglas-Rachford alternatingdirection methods, iterative shrinkage-thresholding algorithms (ISTA),and split inexact Uzawa methods. Examples of the multiplicative updaterules include, but are not limited to, Exponentiated gradient updaterules, and multiplicative updates for positivity constrained quadraticprograms.

In an example, optimizing a pre-diffusion analyte distribution for anexcitation light L5 is implemented in two passes, wherein in a firstpass, all pixels of an image representing the pre-diffusion analytedistribution for the excitation light L5 are considered as potentialanalyte release sites, and in a second pass, by employing at least oneof the aforesaid algorithms/rules, a number of pixels considered aspotential analyte release sites is reduced.

Therefore, it is to be understood that the generated model of analyterelease distribution in the well for each of the plurality of excitationlights is generated in a form of a mathematical function.

In an embodiment, generating the model of analyte release distributionin the well for each of the plurality of excitation lights furthercomprises modifying the generated model of analyte release distributionin the well for each of the at least one excitation light, to analyse atleast one fluorospot therein. Specifically, such modification may relateto gating or pruning of the at least one fluorospot in the generatedmodel of analyte release distribution in the well for each of the atleast one excitation light. Further, such modification may be performedto set ‘cut-offs’ to be employed to exclude and/or include the at leastone fluorospot detected in the generated model of analyte releasedistribution in the well for each of the at least one excitation light.More specifically, such modification may facilitate a user to select theat least one fluorospot for analysis of attributes such as secretionintensity (or amount of analyte secretion) of the cells. For example, bymodifying the generated model of analyte release distribution in thewell for each of the at least one excitation light, the user canseparately study and calculate an amount of the at least one analytesecreted by the cells under investigation.

According to an embodiment, modifying the model of analyte releasedistribution in the well, is based on at least one user-selectableparameter, wherein the at least one parameter is selected from a groupcomprising: fluorospot/fluorescent intensity, fluorospot size, estimatedamount of analyte release, fluorospot colour, area of interest in thewell. Specifically, upon selection of the at least one parameter, theuser may specify a value of the selected at least one parameter as ameasure of sensitivity of fluorospot detection/analysis. In an example,for a given excitation light L6, the user may select a parameter, suchas area of interest in the well, and specify that only fluorospots in aleft half of the well may be analysed. Optionally, in such example, theuser may specify that the fluorospots along a periphery of the well maybe discarded to prevent unwanted artefacts from being analysed. Inanother example, for the given excitation light L6, the user may selecta parameter, such as fluorospot size, and specify that only fluorospotsof radius greater than 30 micrometres may be analysed.

Therefore, it is to be understood that the modified model of analyterelease distribution in the well for each of the plurality of excitationlights is provided in a form of a mathematical function.

Optionally, the method further comprises obtaining the fluorescentintensity at the at least one spatially defined fluorospot. Moreoptionally, the fluorescent intensity at the at least one spatiallydefined fluorospot is obtained based on the generated X-Y table.Specifically, a spatially defined fluorospot is identified from thegenerated X-Y table that emits or radiates fluorescent of highintensity.

The method further comprises clustering a plurality of co-positionedfluorospots as a multiple secretion fluorospot, wherein the clusteringis performed for all generated models of analyte release distribution,and wherein the clustering determines at least one multiple secretionfluorospot. Specifically, all generated models of analyte releasedistribution are processed to identify a plurality of fluorospots ofdifferent analytes that are positioned at a same location (for example,at same pixel coordinates) and/or proximal to each other. Further, insuch instance, the plurality of fluorospots may be considered proximalto each other if their respective locations lie within a predetermineddistance from each other. Specifically, such predetermined distance maybe a maximal distance between fluorospots that can be clusteredtogether. Optionally, such predetermined distance may be specified bythe user. In an embodiment, clustering the plurality of co-positionedfluorospots employs a distance-based hierarchical clustering algorithm.Specifically, the distance-based hierarchical clustering algorithm iscontiguity constrained, thereby, preventing proximal fluorospots of asame generated model of analyte release distribution from beingclustered together.

It is to be understood that the term ‘multiple secretion fluorospot’used herein relates to a secretory footprint of a plurality of analytesthat are secreted from a single cell. Therefore, the plurality ofco-positioned fluorospots for all generated models of analyte releasedistribution, represent the plurality of analytes that are secreted fromthe single cell. Further, the at least one multiple secretion fluorospotdetermined by aforesaid clustering relate to at least one cell thatsecretes the plurality of analytes.

According to an embodiment, the method may further comprise displaying aresultant model of analyte release distribution in the well, wherein theresultant model comprises the detected at least one fluorospot, andwherein single secretion fluorospots and multiple secretion fluorospotsare represented distinctly. Specifically, the resultant model of analyterelease distribution in the well relates to a resultant image that is anoverlay of images of the generated models of analyte releasedistribution, for each of the at least one excitation light. Morespecifically, since the wavelength of fluorospots for different analytesis different, in the resultant model of analyte release distribution inthe well, a wavelength of multiple secretion fluorospots may be acombination of wavelengths of fluorospots of its constituent analytes.For example, there may exist two modified models of analyte releasedistribution in a well to analyse two analytes (such as analytes A3 andA4). In such example, a wavelength of fluorospots of analyte A3 maycorrespond to a green colour, and a wavelength of fluorospots of analyteA4 may correspond to a red colour. Thereafter, in a resultant model ofanalyte release distribution in the well, there exist single secretionfluorospots (of each of the analytes A3 and A4) and multiple secretionfluorospots (of both analytes A3 and A4).

As mentioned previously, the present disclosure describes the system foranalysing fluorospot assays. Specifically, the described system isemployed for implementing the aforementioned method for analysingfluorospot assays.

The system comprises a light source arrangement configured to generateat least one excitation light, wherein at a given time, the light sourcearrangement generates only one excitation light. In an embodiment, thelight source arrangement comprises at least one light source positionedon an actuator arrangement, wherein the at least one light source isimplemented by way of at least one of: Light Emitting Diode, halogenlight source, xenon light source, laser light source. For example, thelight source arrangement may comprise 5 light sources positioned on amotorized actuator arrangement, wherein the motorized actuatorarrangement may be rotatable, tiltable, or displaceable (horizontallyand/or vertically).

Further, the system comprises a collimation arrangement positioned on anoptical path of a generated excitation light, wherein the collimationarrangement is configured to collimate the generated excitation light.Specifically, the collimation arrangement makes rays of the plurality ofexcitation lights parallel to each other. In an example, the collimationarrangement may be at least one collimation lens.

Furthermore, the system comprises a multiband beam splitter positionedon an optical path of the collimated excitation light, the multibandbeam splitter being supported by a support arrangement. The multibandbeam splitter is configured to reflect the collimated excitation lightonto an assay plate for illuminating a well of the assay plate having aplurality of wells, receive a reflection of the excitation light and anemitted light from the assay plate, and reflect a portion of thereflected excitation light whilst transmitting a remaining portion ofthe reflected excitation light and the emitted light to a filteringarrangement. Specifically, the multiband beam splitter receives thereflection of the excitation light mixed with the emitted light from theassay plate, but is unable to completely block the reflection of theexcitation light from passing therethrough. Therefore, the portion ofthe reflected excitation light that is reflected from the multiband beamsplitter relates to a part of the reflected excitation light that isblocked from passing through the multiband beam splitter. Further, theremaining portion of the reflected excitation light that is transmittedthrough the multiband beam splitter relates to remnants of the reflectedexcitation light that do not get blocked from through transmission. Asdescribed previously, the emitted light relates to fluorescence emittedfrom an analyte upon illumination by the collimated excitation light.Therefore, different emitted lights emitted from different analytes arepassed through the multiband beam splitter.

In an embodiment, the assay plate is positioned at a right angle withrespect to the multiband beam splitter. Therefore, the multiband beamsplitter may reflect the collimated excitation light by an angle ofapproximately 90 degrees onto the assay plate for illuminating the wellof the assay plate.

According to an embodiment, the multiband beam splitter is implementedby way of at least one of: a transparent mirror, a semitransparentmirror, a prism, a dichroic mirror. According to another embodiment, thesupport arrangement is a filter cube. Specifically, the filter cube maybe a semitransparent or a transparent cubical structure configured toaccommodate and support the multiband beam splitter therein. Forexample, in the system, a dichoric mirror may be mounted diagonallywithin a filter cube.

The system further comprises the filtering arrangement comprising afilter wheel and at least one emission filter, wherein the filter wheelis configured to accommodate the at least one emission filter therein.The at least one emission filter is configured to remove the remainingportion of the reflected excitation light received from the multibandbeam splitter, and filter the emitted light whilst transmitting theemitted light to an optic arrangement. Specifically, the filter wheel isa rotatable device including at least one housing to accommodate the atleast one emission filter therein. More specifically, the at least oneemission filter may be configured to block light of any wavelength otherthan that of the emitted light.

Further, the system comprises the optical arrangement configured todirect the filtered emitted light onto an imaging device. Specifically,the optical arrangement may include at least one optical element tochange the optical path of the filtered emitted light, so as to focusthe filtered emitted light on to the imaging device. In an embodiment,the optical arrangement comprises at least one of: telecentric lens,macro lens. Optionally, the optical arrangement may also compriseoptical elements such as mirrors, prisms, lenses, and the like.

The system comprises the imaging device configured to capture at leastone image of the well, in raw image format, for each of the at least oneexcitation light. Specifically, the imaging device is selected from anyone of: a high-dynamic-range imaging device, a low-dynamic-range imagingdevice, a digital camera. As mentioned previously, the presentdisclosure employs an HDR imaging device configured to capture the atleast one image of the well, in raw image format, for each of the atleast one excitation light. More specifically, the imaging devicereceives the filtered emitted light from the optical arrangement onto animage sensor of the imaging device, to capture the at least one image.In an embodiment, the imaging device may capture the at least one imagein grayscale. Specifically, higher resolution grayscale images arecaptured by employing raw image format. Beneficially, images captured inraw image format are in unprocessed form as compared to images of otherformats such as Joint Photographic Experts Group (JPEG) format, and aretherefore free from colourspace-dependent encoding. In anotherembodiment, the imaging device may capture the at least one image ascoloured images.

Furthermore, the system comprises a processing module coupled to theimaging device, wherein the processing module is configured to generatethe model of analyte release distribution in the well for each of the atleast one excitation light, and cluster the plurality of co-positionedfluorospots as a multiple secretion fluorospot, wherein the clusteringis performed for all generated models of analyte release distribution,and wherein the clustering determines at least one multiple secretionfluorospot.

Optionally, in this regard, the processing module is further configuredto deconvolve the captured at least one image of the well for the givenexcitation light to estimate a pre-analyte distribution, and detect atleast one potential analyte release site based on local maxima in thepre-diffusion analyte distribution.

Optionally, the system further comprises a tuner, wherein the tuner isoperable to move horizontally along the X-Y plane of the assay plate tothe at least one spatially defined fluorospot, and wherein the sensor isoperable to obtain the fluorescent intensity associated with the atleast one spatially defined fluorospot. Specifically, the tuner visitsevery spatially defined fluorospot on the XY plane of the assay plate.More specifically, the sensor coupled to the tuner records thefluorescent intensity associated with the at least one spatially definedfluorospot. Furthermore, the spatially defined fluorospot with a highfluorescent intensity is counted and its position, as obtained by thegenerated XY table, is provided to one or more components of the systemfor further use, such as data processing.

Optionally, the system further comprises a controller operativelycoupled to the tuner. More optionally, the controller is operable topreset the data corresponding to the well coordinates, depth of well,centre of well and the exact position of the well borders, fluorospotsize, peripheral distance between adjacent wells, distance between thecenters of two adjacent wells and so on, are predefined/preset to fullyautomate the process.

Furthermore, the controller is configured to generate an X-Y tableassociated with movement of the tuner horizontally along the X-Y planeof the assay plate. Specifically, the controller is operable to generatethe X-Y table based on the preset data corresponding to the wellcoordinates, depth of well, centre of well and the exact position of thewell borders, fluorospot size, peripheral distance between adjacentwells, distance between the centers of two adjacent wells and so on. Thecontroller is further operable to provide the generated X-Y table to thetuner, and wherein the tuner is configured to move horizontally alongthe X-Y plane of the assay plate based on the provided X-Y table.Specifically, the generated X-Y table guides the tuner to visit themultiple fluorospots in a predefined linear motion, preferablyhorizontal, over the XY plane of the assay plate. More specifically, thehorizontal movement of the tuner along the X-Y plane of the assay platebased on the provided X-Y table enables assessing all the wells of theassay plate and determining the spatially defined fluorospots with highfluorescent intensity.

Optionally, the controller is operable to generate the X-Y table byemploying a calibration algorithm. As mentioned previously, thecalibration algorithm comprises data corresponding to the wellcoordinates, depth of well, centre of well and the exact position of thewell borders, fluorospot size, peripheral distance between adjacentwells, distance between the centers of two adjacent wells and so onpreset/predefined to fully automate the process. Specifically, thecalibration algorithm governs the movement of tuner over everyfluorospot on the XY plane of the assay plate.

Optionally, the controller is further operable to identify a centre ofthe well. Specifically, the calibration algorithm is developed toidentify the area of interest in the assay plate for detection offluorospots. More specifically, the fluorospots are counted within thearea of interest, and the fluorospots falling outside the area ofinterest are not counted while determining the concentration of at leastone fluorophore-detected analyte in the spatially defined area of thewell. Furthermore, the calibration algorithm has an auto-centeringfunctionality that determines the centers of the well for measurementsof fluorescent intensity. In this case, the calibration algorithmcalculates the distance between the center of two wells to identify thecalibration points. The calibration algorithm moves the tuner to thecalibration points, where the sensor of the tuner measures thefluorescent intensity. In an embodiment, the calibration algorithmcalibrates and recalibrates the X-Y table to avoid the saturation of thesensor over a period of time of use.

Optionally, the controller is further operable to detect a deviation inthe generated X-Y table and the movement of the tuner. Specifically, thecalibration algorithm detects the difference in predefined distance fromthe centers of at least two adjacent wells and the position of thetuner. More specifically, the deviation in the generated X-Y table andthe movement of the tuner is an indication to a wrong calibration. Forexample. If the distance between the center of two adjacent wells (W0and W1) on an assay plate (W) is 1.25 cm, and the same is preset in theX-Y table; however, the tuner moves to a well W1 that is 1 cm away fromthe center of the well W0, in such a case, the controller is operable todetects a deviation of 0.25 cm in the generated X-Y table and themovement of the tuner.

Furthermore, the controller is also operable to provide, based on thedetected deviation, a correction for the movement of the tuner.Specifically, the controller corrects any deviation in the generated X-Ytable and the movement of the tuner. More specifically, the controlleremploys an auto-centering module for the correction of the movement ofthe tuner. More specifically, the auto-centering module corrects themovement of the tuner and places the tuner right above the spatiallydefined fluorospot. For instance, in the above example, for a deviationof 0.25 cm in the generated X-Y table and the movement of the tuner iscorrected by the autocentering module by shifting the tuner right abovethe spatially defined fluorospot.

In an embodiment, the calibration algorithm includes a circular Area ofInterest (AOI) that constrains the area in which spatially definedfluorospots are detected and counted in each image captured. Fluorospotsdetected outside of the AOI are not counted. Specifically, a feature inthe calibration algorithm identifies the well border and automaticallyplaces the AOI in the center position of the well. More specifically, asthe well centers deviates from well-to-well and from plate-to-plate, thewell centering algorithm is absolutely essential in order to generate aconsistent data.

Furthermore, optionally, the processing module is configured to optimizethe pre-diffusion analyte distribution based on the detected at leastone potential release site and modify the generated model of analyterelease distribution in the well for each of the at least one excitationanalyte, to analyse at least one fluorospot therein.

In an embodiment, the system further comprises at least one excitationfilter positioned on an optical path of the collimated excitation light,wherein the at least one excitation filter is configured to filter thecollimated excitation light. Specifically, the at least one excitationfilter may be configured to the wavelengths of the plurality ofexcitation lights such that, at a given time, an excitation filter mayonly pass an excitation light of a desired wavelength whilst blockinglight of other wavelengths from passing therethrough. Therefore, the atleast one excitation filter purifies the collimated excitation light byblocking undesired light components. In such instance, a distinctexcitation filter may be employed for a distinct excitation light.Optionally, the excitation filter may comprise a filter wheel.

In an embodiment, the system may further comprise a visualization modulecoupled to the imaging device and the processing module, wherein thevisualization module comprises a user interface rendered on a computingdevice associated with a user, and wherein the visualization module isconfigured to perform at least of: display the captured at least oneimage for each of the at least one excitation light, display thegenerated model of analyte release distribution in the well for each ofthe at least one excitation light, receive user input to controloperation of the processing module, display a resultant model of analyterelease distribution in the well. Specifically, the visualization modulemay be operable by the user of the system for viewing the analysis ofassays. In an example, the user may modify the generated model ofanalyte release distribution in the well for each of the pluralityofexcitation lights by interacting with the user interface (for example,via touch input, keypad input, mouse input, and the like) to select aparameter such as area of interest in the well, and specify the value ofthe parameter (such as, a left half of the well). Examples of thecomputing device associated with the user include, but are not limitedto, a smartphone, a tablet computer, a desktop computer, a notebookcomputer, and a personal digital assistant.

In an embodiment, the system may further comprise a mask for removingnoise from the at least one excitation light. The term ‘noise’ relatesto random disturbance that influence the overall fluorospot count. Noisemay result from artefacts, such as hair, dust, fibers and the like, thatenter into the wells of the assay plate. Such noise can get counted bythe algorithm or software of the imaging device and result in falsepositive and/or false negatives in the overall fluorospot count. It istherefore necessary to remove the false positives and false negativesone by one, ensuring a noise-free fluorospot count. Noise removal isperformed by adding a layer of paint, such as a mask, to the wellsinfluenced with noise, and is configured to exclude noise behind themask from the overall fluorospot count. Specifically, the term ‘mask’relates to a practical approach for removing noise from an image.Beneficially, the mask excludes such artefacts from the spot-count,wherein all spots behind the mask are excluded from the overallspot-count. Subsequently, the noise is subtracted from the fluorescentintensity to obtain the net fluorescent intensity. The obtained netfluorescent intensity of each well of the assay plate is saved in anyone of image file formats, including, but not limited to, JointPhotographic Experts Group format, Tagged Image File Format, PortableNetwork Graphics format, and Graphics Interchange Format.

For example, when an image of the plate is saved for example in a formatsuch as Joint Photographic Experts Group (JPEG) format, a special JointPhotographic Experts Group (JPEG) image is created, namely Mask (orMask.jpeg) depicting the locations on the well plate where the mask wasadded. Beneficially, a ‘history file’ is created that records eachaction performed over the image. Additionally, history file is used toaccess the possible alterations done on the saved image file.

Furthermore, the mask for removing noise from the plurality ofexcitation lights is operable to be removed from the system arrangementif required. Specifically, the added layer of paint is operable to beremoved, as desired by the user, by simply accessing saved image of theplate and removing the mask to obtain the original data, comprising thefluorospots along with the potential noise associated with thefluorospots. It will be appreciated that in the mask implementation orremoval process, no action or data is ever erased and each action isrecorded in a history file. The history file records each change made inthe process of analysing the FluoroSPot assays.

In yet another aspect, an embodiment of the present disclosure providesa method for analysing spatially defined FluoroSpot assays, the methodcomprising determining the concentration of at least onefluorophore-detected analyte in a spatially defined area of the well;wherein determination of the concentration of the analyte includes:

-   -   (a) identifying the at least one spatially defined fluorospot;    -   (b) sensing the at least one spatially defined fluorospot to        obtain fluorescent intensity associated with the at least one        spatially defined fluorospot; and    -   (c) correlating the obtained fluorescent intensity associated        with the at least one spatially defined fluorospot with the        concentration of at least one fluorophore-detected analyte in        the spatially defined area of the well.

In yet another aspect, an embodiment of the present disclosure providesa system for analysing spatially defined fluorospot assays, the systemcomprising a sensor arrangement to determine the concentration of atleast one fluorophore-detected analyte in a spatially defined area ofthe well, wherein the sensor arrangement comprises:

(a) a sensor operable to read at least one spatially defined fluorospot;and

(b) a tuner configured to obtain fluorescent intensity associated withthe at least one spatially defined fluorospot, characterized in that thetuner is further configured to correlate the obtained fluorescentintensity associated with the at least one spatially defined fluorospotwith the concentration of at least one fluorophore-detected analyte inthe spatially defined area of the well.

Specifically, fluorospot assays can be analyzed for detecting analytessecreted by the cells as well as for determining the concentration of atleast one fluorophore-detected analyte in the spatially defined area ofthe well. Optionally, the analyte includes at least one of: a serumsample, a plasma sample, a urine sample, a liquor sample, a viralparticle, a protein, a cell supernatant, at least one potential analytein the well. Specifically, the analytes include, but are not limited to,cytokines, and immunoglobulins. More specifically, the various samples,such as the serum sample, the plasma sample, the urine sample, theliquor sample, the viral particle, the protein, the cell supernatant,the at least one potential analyte release site in the well, can becultured within the assay plate to increase the concentration of theanalyte secreted by the sample of cells.

The term ‘concentration’ relates to a measurable property. Specifically,the concentration can be measured as mass concentration, volumeconcentration, molar concentration, number concentration, and so forth.Furthermore, the concentration of a solute or solvent in a solution canrange from diluted to concentrated, depending on the amount of thesolute or solvent in the solution. Specifically, the concentration ofanalyte is determined from the model of analyte release distribution inthe well generated for a given excitation light.

The method for determining the concentration of at least onefluorophore-detected analyte in the spatially defined area of the wellcomprises identifying the at least one spatially defined fluorospot. Inan embodiment, the concentration of the at least onefluorophore-detected analyte in the spatially defined area of the wellis based on at least one user-selectable parameter, wherein the at leastone parameter is selected from a group comprising:fluorospot/fluorescent intensity, fluorospot size, estimated amount ofanalyte release, fluorospot colour, area of interest in the well.Specifically, upon selection of the at least one parameter, the user mayspecify a value of the selected at least one parameter as a measure ofsensitivity of fluorospot detection/analysis. More specifically, theconcentration of the at least one fluorophore-detected analyte in thespatially defined area of the well is based on fluorospot/fluorescentintensity. In an example, for the excitation light L7, the user mayselect one or more parameters, such as fluorospot/fluorescent intensity,fluorospot colour and area of interest, and specify that onlyfluorospots with high intensity of green colour are to be selectedwithin the left half of the well.

Further, the method comprises sensing the at least one spatially definedfluorospot to obtain fluorescent intensity associated with the at leastone spatially defined fluorospot. The spatially defined fluorospotprovides a measurable fluorescent signal that is detected by a sensor.Optionally, the concentration of the at least one fluorophore-detectedanalyte distributed in the spatially defined area of the well is basedon fluorescent intensity. It will be appreciated that the concentrationof the fluorophore-detected analyte is proportional to the fluorescenceintensity, and preferably darkness of colour of fluorescence.

Optionally, the method further comprises obtaining the fluorescentintensity at the at least one spatially defined fluorospot. Moreover,obtaining the fluorescent intensity at the at least one spatiallydefined fluorospot includes setting ‘cut-offs’. Specifically, thecut-off is employed to exclude and/or include the at least one spatiallydefined fluorospot detected in the generated model of analyte releasedistribution for each of the at least one excitation light. Moreoptionally, such modification may facilitate sensing the at least onespatially defined fluorospot for analysis of attributes such assecretion intensity and/or concentration of analyte secreted by thecells. Furthermore, such modification may be based on at least oneuser-selectable parameter, wherein the at least one parameter isselected from a group comprising: fluorospot/fluorescent intensity,fluorospot size, estimated amount of analyte release, fluorospot colour,area of interest in the well. Optionally, by combining the fluorescentintensity and capturing images of the well, it is possible to providehigh sensitivity and an excellent measuring range for the concentrationof the fluorophore-detected analyte. Sensing the at least one spatiallydefined fluorospot to obtain fluorescent intensity associated with theat least one spatially defined fluorospot includes sensing thefluorescent intensity at the area of interest in the well, preferably atthe centre. Furthermore, the method further comprises identifying acenter of the well for determining the spatially defined fluorospot. The“center of well” is determined based on the coordinates of the wellobtained by analysing the captured image of the well. Specifically,sensing the at least one fluorospot to obtain fluorescent intensityassociated with the at least one spatially defined fluorospot isperformed automatically, without requiring direct and frequent manualinterventions. More specifically, sensing the at least one fluorospot toobtain fluorescent intensity associated with the at least one fluorospotexcludes the fluorescent intensity associated with the at least onefluorospot that is a result of simple wall reflections. Optionally,sensing the at least one fluorospot to obtain fluorescent intensityassociated with the at least one fluorospot is performed around the areaof interest, preferably a circular area of interest. In an example,fluorospots exhibiting high intensity of green colour are sensed foranalyte A5 in a predefined circular area of interest. It is understoodthat flurospots outside the area of interest are not included in thesensing of at least one fluorospot to obtain fluorescent intensity.

Optionally, the method further comprises generating an X-Y tableassociated with the X-Y plane of the assay plate. In an embodiment, themethod of the present disclosure enables detection of data correspondingto the well coordinates, depth of well, centre of well and the exactposition of the well borders, fluorospot size, peripheral distancebetween adjacent wells, distance from the center of at least twoadjacent wells. Specifically, the method employs an automated machinery,such as a precision motor, that is configured to determine the distancefrom the center of at least two adjacent wells.

Optionally, the method further comprises obtaining the fluorescentintensity at the at least one spatially defined fluorospot. Moreoptionally, the fluorescent intensity at the at least one spatiallydefined fluorospot is obtained based on the generated X-Y table.Specifically, a spatially defined fluorospot is identified from thegenerated X-Y table that emits or radiates fluorescent of highintensity. More specifically, the fluorescence intensity is measured bya fluorescence measuring instrument at the at least one spatiallydefined fluorospot.

Furthermore, the X-Y table is generated by employing a calibrationalgorithm. Specifically, the calibration algorithm is developed tocalibrate the X-Y table. More specifically, the calibration algorithm isdeveloped to calibrate and re-calibrate the X-Y table upon multipleseries of rounds. In an embodiment, the calibration algorithm isprogrammed to move the sensor coupled to the tuner to every fluorospot.In another embodiment, the calibration algorithm is programmed toprotect the sensor from over-current. Optionally, the data correspondingto the well coordinates, depth of well, centre of well and the exactposition of the well borders, fluorospot size, peripheral distancebetween adjacent wells, distance between the centers of two adjacentwells and so on, are predefined/preset in the sensor to fully automatethe process. Alternatively, additionally, the sensor is configured toreceive manual inputs from a user of the fluorescence measuringinstrument.

The method further comprises correlating the obtained fluorescentintensity associated with the at least one spatially defined fluorospotwith the concentration of at least one fluorophore-detected analytedistributed in the spatially defined area of the well. Specifically, theconcentration of the at least one fluorophore-detected analyte in thespatially defined area of the well is proportional to the fluorescentsignal detected by the sensor. The obtained fluorescent intensity may becorrelated with the concentration of at least one fluorophore-detectedanalyte in the spatially defined area of the well. In an embodiment, theconcentration of at least one fluorophore-detected analyte in thespatially defined area of the well is a mathematical function.Furthermore, the mathematical function may represent analyteconcentration in the well. In an example, a net fluorescent intensity offluorospots with a wavelength corresponding to a green colour can becorrelated to an analyte A6 and its concentration in the well. Inanother example, the concentration of A6 in the well is determined asone or more of: mass concentration, volume concentration, molarconcentration, number concentration, and so forth. In yet anotherexample, the concentration of A6 in the well is 3 microgram of analyteA6 per litre of solvent. It is to be understood that although the methoddescribed herein relates to analysis of one well of the assay plate, themethod may be implemented to analyse more than one well of the assayplate. In such instance, two models (G3 and G4) of analyte releasedistribution in a well for two excitation lights (L8 and L9) may begenerated. In such example, the model G3 may represent distribution ofanalyte A7 in the well and the model G4 may represent distribution ofanalyte A8 in the well. Therefore, a wavelength of fluorospots ofanalyte A7 in the model G3 is different from a wavelength of fluorospotsof analyte A8 in the model G4. Furthermore, the concentration C1 of theanalyte A7 in the model G3 is different from the concentration C2 of theanalyte A8 in the model G4.

Optionally, the method further comprises removing noise from the atleast one excitation light. As mentioned previously, noise removal isperformed by employing the mask. Beneficially, the mask excludes suchartefacts from the spot-count, wherein all spots behind the mask areexcluded from the overall spot-count. Subsequently, the noise issubtracted from the fluorescent intensity to obtain the net fluorescentintensity. The obtained net fluorescent intensity of each well of theassay plate is saved in any one of image file formats, including, butnot limited to, Joint Photographic Experts Group format, Tagged ImageFile Format, Portable Network Graphics format, and Graphics InterchangeFormat.

As mentioned previously, the present disclosure describes the system foranalysing fluorospot assays, the system for determining theconcentration of at least one fluorophore-detected analyte in thespatially defined area of the well. Specifically, the determination ofat least one fluorophore-detected analyte requires employing a sensingarrangement to measure the fluorescent intensity of each well, andcorrelating the measured fluorescent intensity of each well with theconcentration of the at least one fluorophore-detected analyte in thespatially defined area of the well. Specifically, the described systemis employed for implementing the aforementioned method for analysingfluorospot assays.

The system for determining the concentration of at least onefluorophore-detected analyte in the spatially defined area of the wellcomprises the sensor operable to read the at least one spatially definedfluorospot. The term ‘sensor’ relates to a high precision measuringinstrument configured to detect fluorescent intensity of varyingwavelengths. Optionally, the sensor is operable to move to at least onefluorospot and determine the centre of the well to detect fluorescentintensity at each well. In an embodiment, the sensor converts thefluorescent intensity associated with the at least one spatially definedfluorospot into an electrical signal for quantifying the fluorescentintensity.

The sensor arrangement further comprises the tuner configured to obtainfluorescent intensity associated with the at least one spatially definedfluorospot. Specifically, the tuner is operatively coupled to the sensorand comprises high precision motors and the calibration algorithm. Morespecifically, the tuner is configured to access the data correspondingto the well coordinates, depth of well, centre of well and the exactposition of the well borders. Subsequent to accessing the suitablefluorospot, the tuner places the sensor at a spatially definedfluorospot, where the sensor detects the at least one spatially definedfluorospot to obtain fluorescent intensity associated with the at leastone spatially defined fluorospot.

Optionally, the tuner is configured to regulate current in the sensorarrangement, preferably in the sensor. Specifically, the tuner preventsthe sensor from over-current induced by the fluorescent intensity whileconverting the fluorescent intensity into the electrical signal.

The tuner is further configured to correlate the obtained fluorescentintensity associated with the at least one spatially defined fluorospotwith the concentration of at least one fluorophore-detected analyte inthe spatially defined area of the well. The net fluorescent intensity isobtained by removing the noise from the obtained fluorescent intensityassociated with the at least one spatially defined fluorospot.Optionally, the tuner is operable to remove noise from the at least oneexcitation light. As mentioned previously, noise removal is performed byemploying the mask. Beneficially, the mask excludes such artefacts fromthe spot-count, wherein all spots behind the mask are excluded from theoverall spot-count. Subsequently, the noise is subtracted from theobtained fluorescent intensity associated with the at least onespatially defined fluorospot to obtain the net fluorescent intensity.The net fluorescent intensity of each well of the assay plate is savedin any one of image file formats, including, but not limited to, JointPhotographic Experts Group format, Tagged Image File Format, PortableNetwork Graphics format, and Graphics Interchange Format. Furthermore,while saving an image of the net fluorescent intensity, a special JointPhotographic Experts Group (JPEG) image is created, namely Mask (orMask.jpeg) depicting the locations on the well plate where the mask wasadded. Additionally, a ‘history file’ is created that records eachaction performed over the image. The history file is used to access thepossible alterations done on the saved image file.

Furthermore, the mask for removing noise from the plurality ofexcitation lights is operable to be removed from the system arrangementif required. Specifically, the added layer of paint is operable to beremoved, as desired by the user, by simply accessing saved image of theplate and removing the mask to obtain the original data, comprising thefluorospots along with the potential noise associated with thefluorospots. It will be appreciated that in the mask implementation orremoval process, no action or data is ever erased and each action isrecorded in a history file. The history file records each change made inthe process of analysing the fluorospot assays.

Optionally, the system further comprises a controller operativelycoupled to the tuner. The controller arrangement is operatively coupledto the tuner to help in determining the concentration of at least onefluorophore-detected analyte in the spatially defined area of the well.Furthermore, the controller is configured to generate an X-Y tableassociated with movement of the tuner horizontally along the X-Y planeof the assay plate. As mentioned previously, the X-Y table enables thetuner to move in a linear, preferably horizontal, direction and assessall the wells of the assay plate and determine the fluorospots with highfluorescent intensity. Optionally, the controller is operable togenerate the X-Y table by employing the calibration algorithm. Thecalibration algorithm enables the controller to generate the X-Y tableby presetting the data corresponding to the well coordinates, depth ofwell, centre of well and the exact position of the well borders,fluorospot size, peripheral distance between adjacent wells, distancefrom the centers of at least two adjacent wells and so on, in thecontroller.

The controller is further operable to provide the generated X-Y table tothe tuner. The tuner is operable to move horizontally along the X-Yplane of the assay plate to the at least one spatially definedfluorospot, and wherein the sensor is operable to obtain the fluorescentintensity associated with the at least one spatially defined fluorospot.As mentioned previously, the X-Y table is generated by employing acalibration algorithm. The tuner is configured to move horizontallyalong the X-Y plane of the assay plate based on the provided X-Y tablebased on the calibration algorithm. The horizontal movement of the tuneralong the X-Y plane of the assay plate based on the provided X-Y tableenables assessing all the wells of the assay plate and determining thespatially defined fluorospots with high fluorescent intensity.

Optionally, the controller is further operable to identify a centre ofthe well. Specifically, the calibration algorithm is developed toidentify the area of interest in the assay plate for detection offluorospots. More specifically, the fluorospots are counted within thearea of interest, and the fluorospots falling outside the area ofinterest are not counted while determining the concentration of at leastone fluorophore-detected analyte in the spatially defined area of thewell. Furthermore, the calibration algorithm has an auto-centeringfunctionality that determines the centers of the well for measurementsof fluorescent intensity. In this case, the calibration algorithmcalculates the distance between the center of two wells to identify thecalibration points. The calibration algorithm moves the tuner to thecalibration points, where the sensor of the tuner measures thefluorescent intensity. In an embodiment, the calibration algorithmcalibrates and recalibrates the X-Y table to avoid the saturation of thesensor over a period of time of use.

Optionally, the controller is further operable to detect a deviation inthe generated X-Y table and the movement of the tuner. Specifically, thecalibration algorithm detects the difference in predefined distance fromthe centers of at least two adjacent wells and the position of thetuner. More specifically, the deviation in the generated X-Y table andthe movement of the tuner is an indication to a wrong calibration.

However, the controller is further operable to provide, based on thedetected deviation, a correction for the movement of the tuner.Specifically, the controller possesses an auto-centering module for thecorrection of the movement of the tuner. More specifically, theauto-centering module corrects the movement of the tuner and places thetuner right above the spatially defined fluorospot. In an embodiment,the calibration algorithm includes a circular Area of Interest (AOI)that constrains the area in which spatially defined fluorospots aredetected and counted in each image captured. Fluorospots detectedoutside of the AOI are not counted. Specifically, a feature in thecalibration algorithm identifies the well border and automaticallyplaces the AOI in the center position of the well. More specifically, asthe well centers deviates from well-to-well and from plate-to-plate, thewell centering algorithm is absolutely essential in order to generate aconsistent data. In an embodiment, the tuner may comprise an in-builtsensor that is configured to detect the fluorospot/fluorescent intensityand convert it to the electrical signal corresponding to thefluorospot/fluorescent intensity.

In a yet another aspect, embodiments of the present disclosure provide asoftware product recorded on machine-readable non-transitory(non-transient) data storage media, wherein the software product isexecutable upon computing hardware for implementing the aforementionedmethod; in other words, the present disclosure provides a computerprogram product comprising a non-transitory computer-readable storagemedium having computer-readable instructions stored thereon, thecomputer-readable instructions being executable by a computerized devicecomprising processing hardware to execute a method for analysingfluorospot assays.

DETAILED DESCRIPTION OF THE DRAWINGS

Referring to FIG. 1 , illustrated is a schematic illustration of asystem 100 for analysing assays, in accordance with an embodiment of thepresent disclosure. The system 100 includes a light source arrangement102 configured to generate at least one excitation light, wherein at agiven time, the light source arrangement 102 generates only oneexcitation light. As shown, the light source arrangement 102 includes atleast one light source, depicted as light sources 104, 106, and 108positioned on an actuator arrangement 110. For example, light sources104-108 are implemented by way of Light Emitting Diodes. It is to beunderstood, that an excitation light generated from the light source 106is depicted as dashed lines, and an optical path of the excitation lightis depicted by use of arrows along the dashed lines. The system 100includes a collimation arrangement 112 positioned on an optical path ofthe generated excitation light to collimate the generated excitationlight, and may contain at least one excitation filter, depicted asexcitation filter 114, to filter the collimated excitation light.

As shown, the system 100 includes a multiband beam splitter 116positioned on an optical path of the filtered excitation light, themultiband beam splitter 116 being supported by a support arrangement118. For example, the multiband beam splitter 116 is implemented by wayof a dichroic mirror and the support arrangement 118 is a filter cube.As shown, the multiband beam splitter 116 reflects the filteredexcitation light onto an assay plate 120 for illuminating a well of theassay plate 120 having a plurality of wells. The multiband beam splitter116 is also configured to receive a reflection of the excitation lightand an emitted light from the assay plate and reflect a portion of thereflected excitation light whilst transmitting a remaining portion ofthe reflected excitation light and the emitted light to a filteringarrangement 122. It is to be understood that a combination of thereflection of the excitation light and the emitted light, is depicted asa dashed-dot line having arrows (commonly known as a centre line).

The filtering arrangement 122 includes a filter wheel 124 and at leastone emission filter (depicted as emission filters 126 and 128), whereinthe filter wheel 124 is configured to accommodate the at least oneemission filter 126-128 therein. As shown, the emission filter 126 isconfigured to remove the remaining portion of the reflected excitationlight received from the multiband beam splitter 116 and filter theemitted light whilst transmitting the emitted light to an opticarrangement 130. It is to be understood that the filtered emitted lightis depicted as a dotted line and an optical path of the filtered emittedlight is depicted by use of arrows along the dotted lines. The opticalarrangement 130 is configured to direct the filtered emitted light ontoan imaging device 132. The system 100 further includes a processingmodule (not shown) coupled to the imaging device 132.

Referring to FIGS. 2A, 2B and 2C, illustrated are schematicillustrations of images of a generated model of a first analyte releasedistribution, a generated model of a second analyte releasedistribution, and a resultant model of analyte release distribution in awell 200, respectively, in accordance with an embodiment of the presentdisclosure.

With reference to FIG. 2A, illustrated is an image of the generatedmodel of the first analyte release distribution in the well 200, inaccordance with an embodiment of the present disclosure. As shown, aplurality of fluorospots 202A, 202B, 202C, and 202D of the first analyteare depicted as crosses.

With reference to FIG. 2B, illustrated is an image of the generatedmodel of the second analyte release distribution in the well 200, inaccordance with an embodiment of the present disclosure. As shown, aplurality of fluorospots 204A, 204B, 204C, 204D, and 204E of the secondanalyte are depicted as dots.

With reference to FIG. 2C, illustrated is an image of the resultantmodel of analyte release distribution in the well 200, in accordancewith an embodiment of the present disclosure. As shown, FIG. 2C is anoverly of images of FIGS. 2A and 2B, wherein the plurality offluorospots 202A-D of the first analyte and the plurality of fluorospots204A-E of the second analyte are depicted distinctly. As shown, thefluorospots 202A and 204A are co-positioned and are therefore determinedas a first multiple secretion fluorospot. Similarly, the fluorospots202C and 204D are co-positioned and are therefore determined as a secondmultiple secretion fluorospot.

FIGS. 2A, 2B and 2C are merely examples, which should not unduly limitthe scope of the claims herein. A person skilled in the art willrecognize many variations, alternatives, and modifications ofembodiments of the present disclosure.

Referring to FIG. 3 , illustrated are steps of a method 300 foranalysing assays, in accordance with an embodiment of the presentdisclosure. At step 302, a well of an assay plate having plurality ofwells, is illuminated with at least one excitation light, wherein at agiven time, the well of the assay plate is illuminated by only oneexcitation light. At step 304, at least one image of the well, in rawimage format, is captured for each of the at least one excitation light.At step 306, a model of analyte release distribution in the well isgenerated for each of the at least one excitation light. The generationof the model of analyte release distribution in the well for a givenexcitation light comprises deconvolving the captured at least one imageof the well for the given excitation light to estimate a pre-diffusionanalyte distribution, wherein such deconvolution is performed prior tolocal diffusion of at least one analyte, and detecting at least onepotential analyte release site based on local maxima in thepre-diffusion analyte distribution. At step 308, a plurality ofco-positioned fluorospots are clustered as a multiple secretionfluorospot, wherein the clustering is performed for all generated modelsof analyte release distribution, and wherein the clustering determinesat least one multiple secretion fluorospot.

The steps 302 to 308 are only illustrative and other alternatives canalso be provided where one or more steps are added, one or more stepsare removed, or one or more steps are provided in a different sequencewithout departing from the scope of the claims herein.

Referring to FIG. 4 , illustrated is a schematic illustration of asystem 400 for analysing spatially defined fluorospot assays, inaccordance with an embodiment of the present disclosure. The system 400comprises a sensor arrangement 402 to determining the concentration ofat least one fluorophore-detected analyte in the spatially defined areaof the well. Furthermore, the sensor arrangement 402 comprises a sensor404 operable to read at least one fluorospot. The sensor arrangement 402further comprises a tuner arrangement 406 configured to obtainfluorescent intensity associated with the at least one spatially definedfluorospot. The tuner arrangement 406 is further configured to correlatethe obtained fluorescent intensity associated with the at least onespatially defined fluorospot with the concentration of at least onefluorophore-detected analyte in the spatially defined area of the well.

Referring to FIG. 5 , illustrated are steps of a method 500 foranalysing spatially defined fluorospot assays, in accordance with anembodiment of the present disclosure. The method 500 comprisesdetermining the concentration of at least one fluorophore-detectedanalyte in the spatially defined area of the well. At step 502, the atleast one spatially defined fluorospot is identified. At step 504, theat least one spatially defined fluorospot is sensed to obtainfluorescent intensity associated with the at least one spatially definedfluorospot. At step 506, the obtained fluorescent intensity associatedwith the at least one spatially defined fluorospot is correlated withthe concentration of at least one fluorophore-detected analyte in thespatially defined area of the well.

The steps 502 to 508 are only illustrative and other alternatives canalso be provided where one or more steps are added, one or more stepsare removed, or one or more steps are provided in a different sequencewithout departing from the scope of the claims herein.

Modifications to embodiments of the present disclosure described in theforegoing are possible without departing from the scope of the presentdisclosure as defined by the accompanying claims. Expressions such as“including”, “comprising”, “incorporating”, “have”, “is” used todescribe and claim the present disclosure are intended to be construedin a non-exclusive manner, namely allowing for items, components orelements not explicitly described also to be present. Reference to thesingular is also to be construed to relate to the plural.

The invention claimed is:
 1. A method for analysing fluorospot assays, the method comprising: (i) illuminating a well of an assay plate having a plurality of wells, with a plurality of excitation lights, wherein at a given time, the well of the assay plate is illuminated by only one excitation light; (ii) capturing at least one image of the well, in raw image format, for each of the plurality of excitation lights; (iii) generating a model of analyte release distribution in the well for each of the plurality of excitation lights, wherein the generation of the model of analyte release distribution in the well for a given excitation light comprises: (a) deconvolving the captured at least one image of the well for the given excitation light to estimate a pre-diffusion analyte distribution; and (b) detecting at least one potential analyte release site based on local maxima in the pre-diffusion analyte distribution; and (iv) for the plurality of excitation lights employed to illuminate the well of the assay plate, clustering a plurality of co-positioned fluorospots as a multiple secretion fluorospot, wherein the clustering is performed for all generated models of analyte release distribution, and wherein the clustering determines at least one multiple secretion fluorospot, wherein the method includes: generating the model of analyte release distribution in the well for each of the plurality of excitation lights comprises optimizing the pre-diffusion analyte distribution based on the detected at least one potential release site; and modifying the model of analyte release distribution in the well to analyse at least one fluorospot therein, wherein optimizing the pre-diffusion analyte distribution, based on the detected at least one potential analyte release site, is implemented iteratively at least once, and comprises employing at least one of: alternating direction methods, multiplicative update rules, forward-backward proximal gradient algorithms.
 2. A method according to claim 1, wherein the plurality of excitation lights for illuminating the well of the assay plate depends on at least one fluorophore-detected analyte distributed across the well.
 3. A method according to claim 1, wherein modifying the model of analyte release distribution in the well is based on at least one user-selectable parameter, wherein the at least one parameter is selected from a group comprising: fluorospot intensity, fluorospot size, estimated amount of analyte release, fluorospot colour, area of interest in the well.
 4. A method according to claim 1, wherein modifying the model of analyte release distribution in the well is based on at least one user-selectable parameter, wherein the at least one parameter is selected from a group comprising: fluorospot intensity, fluorospot size, estimated amount of analyte release, fluorospot colour, area of interest in the well.
 5. A method according to claim 1, wherein clustering the plurality of co-positioned fluorospots employs a distance-based hierarchical clustering algorithm.
 6. A method according to claim 1, further comprising displaying a resultant model of analyte release distribution in the well, wherein the model of analyte release distribution, based on the detected at least one potential analyte release site, is implemented iteratively at least once, and comprises employing at least one of: alternating direction methods, multiplicative update rules, forward-backward proximal gradient algorithms, wherein the resultant model comprises the detected at least one fluorospot, and wherein single secretion fluorospots and multiple secretion fluorospots are represented.
 7. A system for analysing fluorospot assays, the system comprising: (i) a light source arrangement configured to generate a plurality of excitation lights, wherein at a given time, the light source arrangement generates only one excitation light; (ii) a collimation arrangement positioned on an optical path of a generated excitation light, wherein the collimation arrangement is configured to collimate the generated excitation light; (iii) a multiband beam splitter positioned on an optical path of the collimated excitation light, the multiband beam splitter being supported by a support arrangement, wherein the multiband beam splitter is configured to: (a) reflect the collimated excitation light onto an assay plate for illuminating a well of the assay plate having a plurality of wells; (b) receive a reflection of the excitation light and an emitted light from the assay plate; and (c) reflect a portion of the reflected excitation light whilst transmitting a remaining portion of the reflected excitation light and the emitted light to a filtering arrangement; (iv) the filtering arrangement comprising a filter wheel and at least one emission filter, wherein the filter wheel is configured to accommodate the at least one emission filter therein, and wherein the at least one emission filter is configured to (d) remove the remaining portion of the reflected excitation light received from the multiband beam splitter; and (e) purify the emitted light whilst transmitting the emitted light to an optic arrangement; (v) the optical arrangement configured to direct the purified emitted light onto an imaging device; (vi) the imaging device configured to capture at least one image of the well, in raw image format, for each of the plurality of excitation light; and (vii) a processing module coupled to the imaging device, wherein the processing module is configured to (f) generate a model of analyte release distribution in the well for each of the plurality of excitation lights by optimizing a pre-diffusion analyte distribution based on the detected at least one potential release site; wherein optimizing the pre-diffusion analyte distribution is implemented iteratively at least once, and comprises employing at least one of: alternating direction methods, multiplicative update rules, forward-backward proximal gradient algorithms; and (g) for the plurality of excitation lights employed to illuminate the well of the assay plate, cluster a plurality of co-positioned fluorospots as a multiple secretion fluorospot, wherein the clustering is performed for all generated models of analyte release distribution, and wherein the clustering determines at least one multiple secretion fluorospot.
 8. A system according to claim 7, wherein the system further comprises at least one excitation filter positioned on an optical path of the collimated excitation light, and wherein the at least one excitation filter is configured to purify the collimated excitation light.
 9. A system according to claim 7, wherein the light source arrangement comprises at least one light source positioned on an actuator arrangement, wherein the at least one light source is implemented by way of at least one of: Light Emitting Diode, halogen light source, xenon light source, laser light source.
 10. A system according to claim 7, wherein the multiband beam splitter is implemented by way of at least one of: a transparent mirror, a semi-transparent mirror, a prism, a dichroic mirror.
 11. A system according to claim 7, wherein the optical arrangement comprises at least one of: telecentric lens, macro lens.
 12. A system according to claim 7, wherein the support arrangement is a filter cube.
 13. A system according to claim 7, further comprising a visualization module coupled to the imaging device and the processing module, wherein the visualization module comprises a user interface rendered on a computing device associated with a user, and wherein the visualization module is configured to perform at least of: display the captured at least one image for each of the at least one excitation light, display the generated model of analyte release distribution in the well for each of the at least one excitation light, receive user input to control operation of the processing module, display a resultant model of analyte release distribution in the well. 